Self-assembly and aggregation of glucagon-like peptide 1 and its analogues

Change log
Přáda, Eva 

Aggregation and physical instability of peptide-based drugs poses a great challenge to the pharmaceutical industry. Glucagon-like peptide 1 (GLP-1) is a hormone that is used in the treatment of type-2 diabetes. However, GLP-1 has a short half-life in vivo and it is prone to aggregate which complicates its pharmaceutical usage. Strategies to overcome the short half-life in vivo include numerous chemical modifications of the native peptide. The focus of this Thesis is on the effect of two sets of chemical modification strategies, lipidation and C-terminal amidation, on the physical stability of the peptide.

The first part of this work combines experimental and computational approaches to better understand the molecular basis of the aggregation of GLP-1 and its C-terminally amidated variant, GLP-1-Am. In particular, the off-pathway aggregation of GLP-1 and GLP-1-Am into disordered low-molecular weight oligomers is described. This process competes with the amyloid formation pathway and the addition of pre-formed off-pathway oligomers slightly slows down the fibrillation rate. Energy Landscape Theory was employed to investigate and rationalize the conformational behaviour and aggregation propensity of GLP-1 in different protonation states. Under all conditions studied, the GLP-1 energy landscape possesses a multi-funnel character with a variety of structurally different ensembles with low energy, which is a typical feature of intrinsically disordered proteins and aggregating systems. It is also shown that β-structure-containing conformations are more energetically favoured at acidic pH compared to neutral pH conditions, which agrees with a greater propensity of GLP-1 for aggregation at acidic pH which was observed experimentally.

The second part of this Thesis focuses on the self-assembly and aggregation of lipidated analogues of GLP-1. Four lipidated GLP-1 analogues, which varied in the position of lipidation, and one additional analogue differing by the nature of the lipid moiety, were studied to establish the effect of the lipidation site and the lipid moiety on the physical stability of the peptide. The lipidation was shown to induce formation of large stable oligomers (i.e. > 7 monomeric units). The aggregation mechanism and kinetics were shown to be highly dependent on the lipidation position and the nature of the lipid moiety. Moreover, the aggregation kinetics of lipidated analogues were rarely observed to follow a classical nucleation-elongation mechanism but were rather likely to consist of more complex processes. Aggregates with a high content of β-sheet were formed by all analogues studied, however, they were distinct in their tertiary structure and aggregate morphology.

Jackson, Sophie
aggregation, amyloid, fibril, GLP-1, oligomer, peptide, self-assembly
Doctor of Philosophy (PhD)
Awarding Institution
University of Cambridge
Peterhouse Cambridge Studentship